Metallo-β-lactamase: structure and mechanism

Wang, Zhigang; Fast, Walter; Valentine, Ann M.; Benkovic, Stephen J.

doi:10.1016/s1367-5931(99)00017-4

Cited by 289 publications

(301 citation statements)

References 49 publications

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“…7 However, it has also been revealed that major differences in the coordination states of the metal ions do exist: some enzymes have two high-affinity zinc-binding sites, whereas other metallo-b-lactamase orthologs have one high-and one low-affinity zinc-binding site. 14 YcbL has been identified as a member of the metallo-b-lactamase family. YcbL from Salmonella enterica serovar Typhimurium has been annotated as a metallo-b-lactamase in the NCBI database while certain orthologs have been designated as GLX2s with putative binding of Zn 2þ , Mn 2þ , and Fe 2þ .…”

Section: Introductionmentioning

confidence: 99%

Structural and functional characterization of Salmonella enterica serovar Typhimurium YcbL: An unusual Type II glyoxalase

et al. 2010

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YcbL has been annotated as either a metallo-b-lactamase or glyoxalase II (GLX2), both members of the zinc metallohydrolase superfamily, that contains many enzymes with a diverse range of activities. Here, we report crystallographic and biochemical data for Salmonella enterica serovar Typhimurium YcbL that establishes it as GLX2, which differs in certain structural and functional properties compared with previously known examples. These features include the insertion of an ahelix after residue 87 in YcbL and truncation of the C-terminal domain, which leads to the loss of some recognition determinants for the glutathione substrate. Despite these changes, YcbL has robust GLX2 activity. A further difference is that the YcbL structure contains only a single bound metal ion rather than the dual site normally observed for GLX2s. Activity assays in the presence of various metal ions indicate an increase in activity above basal levels in the presence of manganous and ferrous ions. Thus, YcbL represents a novel member of the GLX2 family.

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Section: Introductionmentioning

confidence: 99%

Structural and functional characterization of Salmonella enterica serovar Typhimurium YcbL: An unusual Type II glyoxalase

et al. 2010

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“…Subclass B3 enzymes exhibit a broad spectrum activity profile with a putative preference for cephalosporins (5). The question as to whether zinc-␤-lactamases are active as the mono-or the dizinc enzyme has been controversially discussed, and two alternative mechanisms for both enzyme states have been proposed (6,7).…”

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Substrate-activated Zinc Binding of Metallo-β-lactamases

Wommer¹,

Rival²,

Heinz³

et al. 2002

Journal of Biological Chemistry

116

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We have investigated the influence of substrate binding on the zinc ion affinity of representatives from the three metallo-␤-lactamase subclasses, B1 (BcII from Bacillus cereus and BlaB from Chryseobacterium meningosepticum), B2 (CphA from Aeromonas hydrophila), and B3 (L1 from Stenotrophomonas maltophilia). By competition experiments with metal-free apoenzymes and chromophoric zinc chelators or EDTA, we determined the dissociation constants in the absence and presence of substrates. For the formation of the monozinc enzymes we determined constants of 1.8, 5.1, 0.007, and 2.6 nM in the absence and 13.6, 1.8, 1.2, and 5.7 pM in the presence of substrates for BcII, BlaB, CphA, and L1, respectively. A second zinc ion binds in the absence (presence) of substrates with considerably higher dissociation constants, namely 1.8 (0.8), 0.007 (0.025), 50 (1.9), and 0.006 (0.12) M for BcII, BlaB, CphA, and L1, respectively. We have concluded that the apo form might be the prevailing state of most of the metallo-␤-lactamases under physiological conditions in the absence of substrates. Substrate availability induces a spontaneous self-activation due to a drastic decrease of the dissociation constants, resulting in the formation of active mononuclear enzymes already at picomolar free zinc ion concentrations. In the presence of substrates, the binuclear state of the enzymes only exists at unphysiologic high zinc concentrations and might be of no biological relevance. From the competition experiments with EDTA it is further concluded that the reactivation rate does not depend on the pool of free zinc ions but proceeds via the EDTA-Zn(II)-enzyme ternary complexes.Metallo-␤-lactamases (class B ␤-lactamases) are produced by bacteria as extracellular or periplasmatic enzymes. All known representatives possess two conserved metal binding sites and require zinc ions as enzymatic cofactors. By catalyzing the hydrolysis of ␤-lactams they render the corresponding strains resistant to almost all ␤-lactam antibiotics. Their increasing emergence in pathogenic bacterial strains due to a rapid dissemination by horizontal gene transfer has induced a growing interest in this enzyme family because of the lack of efficient therapies to treat infected patients.The metallo-␤-lactamases constitute a group of heterogeneous proteins that is divided into subclasses B1, B2, and B3 (1). Subclass B1 exhibits a broad substrate profile (2), and its zinc binding sites are composed of His-116, His-118, and His-196 (site 1) and Asp-120, Cys-221, and His-263 (site 2) (numbering according to Ref. 3). In subclass B2 the zinc ligands in site 2 are conserved, whereas His-116 in site 1 is replaced by Asn. Representatives of subclass B2 efficiently hydrolyze only carbapenems (2) and are active as monozinc enzymes, whereas the binding of a second zinc ion causes non-competitive inhibition (4). In subclass B3, Cys-221 is substituted by a Ser and is replaced by His-121 as a zinc ligand in site 2. In the Gob-1 enzyme (Chryseobacterium meningosepticum PINT) an additional H...

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“…Enzymes in classes A, C, and D utilize an active site serine in the covalent catalysis of the ␤-lactam hydrolysis, whereas class B consists of metalloenzymes with one or two zinc cofactors. Although the catalytic mechanism of the serine-based enzymes is relatively well established (2), our understanding of class B ␤-lactamases is less developed (4).…”

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Catalytic Mechanism of Class B2 Metallo-β-lactamase

Xie

Guo

2006

Journal of Biological Chemistry

105

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The initial nucleophilic substitution step of biapenem hydrolysis catalyzed by a subclass B2 metallo-␤-lactamase (CphA from Aeromonas hydrophila) is investigated using hybrid quantum mechanical/molecular mechanical methods and density functional theory. We focused on a recently proposed catalytic mechanism that involves a non-metal-binding water nucleophile in the active site of the monozinc CphA. Both theoretical models identified a single transition state featuring nearly concomitant nucleophilic addition and elimination steps, and the activation free energy from the potential of mean force calculations was estimated to be ϳ14 kcal/ mol. The theoretical results also identified the general base for activating the water nucleophile to be the metal-binding Asp-120 rather than His-118, as suggested earlier. The protonation of Asp-120 leads to cleavage of the O ␦2 -Zn coordination bond, whereas the negatively charged nitrogen leaving group resulting from the ring opening replaces Asp-120 as the fourth ligand of the sole zinc ion. The electrophilic catalysis by the metal ion provides sufficient stabilization for the leaving group to avoid a tetrahedral intermediate. The theoretical studies provided detailed insights into the catalytic strategy of this unique metallo-␤-lactamase.The excessive use and abuse of ␤-lactam antibiotics have accelerated the spread of drug-resistant bacterial strains. The unprecedented level of antibiotic resistance threatens to destroy their efficacy in treating infectious diseases, posing a grand challenge to public health (1). The primary defense strategy adopted by bacteria is to deactivate the antibiotics by hydrolytic cleavage of the ␤-lactam ring, catalyzed by ␤-lactamases (2). These enzymes can be divided into four classes (3). Enzymes in classes A, C, and D utilize an active site serine in the covalent catalysis of the ␤-lactam hydrolysis, whereas class B consists of metalloenzymes with one or two zinc cofactors. Although the catalytic mechanism of the serine-based enzymes is relatively well established (2), our understanding of class B ␤-lactamases is less developed (4).Metallo-␤-lactamases often have very broad substrate spectra (5) stemming apparently from the metal-dependent catalytic mechanism. Despite much effort, no clinically effective inhibitor has been found. On the other hand, increasing evidence in recent years has pointed to rapid proliferation of these metalloenzymes in pathogenic microorganisms (6). Hence, these enzymes represent a potentially more potent threat to the existing arsenal of ␤-lactam antibiotics than other classes of ␤-lactamases (5, 6).Class B ␤-lactamases can be further separated into three subclasses. Despite considerable sequence diversity (4), their catalytic scaffolds are relatively conserved. All class B ␤-lactamases have two potential metal binding sites (7-11). Protein ligands in the so-called Zn1 site include three His residues in the B1 and B3 subclasses, but a His residue is replaced by Asn in B2 subclass ␤-lactamases. The Zn2 site has an Asp...

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Metallo-β-lactamase: structure and mechanism

Cited by 289 publications

References 49 publications

Structural and functional characterization of Salmonella enterica serovar Typhimurium YcbL: An unusual Type II glyoxalase

Structural and functional characterization of Salmonella enterica serovar Typhimurium YcbL: An unusual Type II glyoxalase

Substrate-activated Zinc Binding of Metallo-β-lactamases

Catalytic Mechanism of Class B2 Metallo-β-lactamase

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